The objective of this proposal is to define the molecular mechanisms of lung alveolar type I cell development, focusing on one of the principal features of type I cells, their extensive, thin, flat shape. In late gestation, precursor distal lung epithelial cells change their shape from cuboidal to flat, acquiring the structural and molecular features of differentiated type I cells to form the thin alveolar gas exchange surface. We hypothesize that genes that regulate plasma membrane growth and polarization, and cytoskeletal organization play a critical inductive and/or permissive role in the process of late fetal type I cell development. We will analyze three important features of alveolar type I cells: shape, type I specific gene expression, and flattening-related gene expression. We will selectively modify each of these features and study alterations in the other two. This approach will provide important information about the molecular mechanisms that initiate and/or sustain type I cell morphogenesis. We will study in the developing lung the roles of genes associated with epithelial cell expansion and flattening identified in Drosophila and C. elegans, and of genes associated with altered cell shape in T1a null mutant mouse, where type I cell formation is impaired. We will determine when and where these genes are expressed in normal lung. We will evaluate their role in cell flattening and spreading using type l-precursor cells isolated at different developmental stages from fetal lungs expressing GFP driven by the promoter of the type I cell gene T1a. We will increase or reduce expression of selected genes in vitro to determine effects on epithelial cell flattening and spreading and on type I specific gene expression. We will modulate the shape of epithelial cells in vitro using culture conditions that restrict spreading and evaluate type I specific and cell-flattening-related gene expression. Finally, we will evaluate the role of these genes in vivo using developing lungs with impaired type I cell differentiation. Analysis of type I cells by these approaches will provide new insights into the regulation of type I cell formation in the fetal lung. This regulation is likely important for type I cell morphogenesis in postnatal lung growth and in lung repair after injury in the adult lung. Relevance to Public Health: When lung development is delayed or babies are delivered prematurely the cells that line the lung alveoli are immature and cannot efficiently perform the normal process of gas exchange. Identifying the key genes that control alveolar cell formation is important to allow the design of new treatments to stimulate newborn lung maturation. Similar mechanisms could apply to the process of alveolar cell healing after injuries caused by infections or environmental factors. Therefore these studies will provide new understanding of the regulation of type I formation that likely will improve treatment of acute and chronic lung diseases in the adult.